PROJECT SUMMARY/ABSTRACT Three major contributors to therapeutic resistance that have been difficult to overcome in pancreatic cancer (PDAC) are mutations in the KRAS oncogene, the presence of a dense desmoplastic stroma that acts as a barrier to drug delivery and effector immune cell infiltration, and the immunosuppressive tumor microenvironment (TME) that renders the tumor ineffective to immunotherapy. Our efforts at targeting downstream effectors of oncogenic RAS, have shown that MEK inhibition (MEKi) results in reciprocal activation of STAT3 signaling, which confers therapeutic resistance and continued PDAC cell growth. Combined inhibition of JAK/STAT3 (STAT3i) and MEKi overcomes therapeutic resistance following RAS inhibition that is mediated through parallel feedback loop activation. We have now identified a novel mechanism showing that combined MEKi and STAT3i also inhibits tumor fibrosis and enhances CD8+ cytotoxic T cell (CTL) infiltration to the tumor while downregulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) in the TME, resulting in reduced tumor burden and improved survival in genetically engineered mouse models (GEMs) of PDAC. In addition, we show that the tumor suppressive effects of MEKi and STAT3i are T cell dependent. This change in the TME, however, is accompanied by sustained PD-L1/PD-1 and CTLA-4 expression. Our preliminary results further show that combined MEKi and STAT3i with PD-1 inhibition can harness the effects of immune checkpoint inhibitors for an enhanced anti-tumor response. Therapeutic strategies that reprogram the tumor stroma to activate T-cell anti-tumor immunity and reverse immune tolerance are of paramount importance as they have the potential to revolutionize treatment for pancreatic cancer and improve clinical outcomes. Our central hypothesis is that MEKi and STAT3i will reprogram cellular components of the PDAC TME to stimulate infiltration of CD8+ CTLs and overcome the immunosuppressive milieu of PDAC to enhance the effects of checkpoint inhibition (CPI) for a durable and sustained anti-tumor response. This will be proven by the following specific aims: Aim 1: Determine if checkpoint inhibition with MEKi and STAT3i will improve survival in GEMs of PDAC. Safety and efficacy of MEKi/STAT3i and anti-PD1 and/or anti-CTLA-4 antibodies treatment response will be determined in two different GEMs of PDAC. Aim 2: Determine if changes in the stromal and immune microenvironment induced by MEKi/STAT3i and checkpoint inhibition result in a durable and sustained anti-tumor immune response in PDAC in vivo. In this aim, multiplex flow cytometry will be used to detect the changes in the cell types and activation phenotypes to determine if the differences pre- and post-treatment predict response. Aim 3: Determine the effects of PDAC cell specific and CAFs specific knockdown of MEK and/or STAT3 signaling on changes in the stromal and immune microenvironment in PDAC. This aim will elucidate the mechanism of cell-specific knockdown of MEK and/or STAT3 mediated changes in that result in increased infiltration CD8+ T cells and suppression of suppressive MDSCs and Tregs. This work will not only evaluate a novel treatment strategy for PDAC, but may uncover potential biomarkers of response to checkpoint inhibitors.